【摘要】：生物礦化是指生物體在有機基質(zhì)的參與和調(diào)控下,形成無機固相礦物的過程。此過程中蛋白分子的添加,可以有效的促進或者抑制生物的無機化進程。本研究首先通過凝膠法合成了可用于原子力顯微鏡(AFM)實驗的一水合草酸鈣(COM)晶體,再通過體外合成不同磷酸化程度的收鈣素(CS)多肽片段,結(jié)合鈣離子電極檢測及原位原子力顯微鏡(AFM)等物理化學表征手段,模擬了收鈣素對COM晶體成核及表面生長的影響,得到的主要結(jié)果如下:(1)選用硅膠和膨潤土作為培養(yǎng)介質(zhì),凝膠法合成COM晶體結(jié)果表明,在硅膠體系中Ca2+和C2O42-濃度為0.5 mol·L-1,室溫培養(yǎng)60天;膨潤土體系中,Ca2+和C2O42-濃度為0.75 mol·L-1室溫培養(yǎng)90天和濃度為0.5 mol·L-1,62℃培養(yǎng)60天條件下合成的COM晶體結(jié)構(gòu)穩(wěn)定,表面平整,尺寸合適(晶面寬度不小于20μm),基本滿足AFM下的樣品實驗要求。(2)在近生理條件下(σCOM=1.62,I=0.15 mol·L-1,p H=7.0,25°C),借助鈣電極監(jiān)控的方法,研究在不同本底電解質(zhì)(KCl或Na Cl)下,含有14個氨基酸殘基的CS多肽(14-mer CS)對COM成核和生長的影響。結(jié)果表明,在KCl作為本底電解質(zhì)的溶液體系中,COM成核速度高于Na Cl中,添加非磷酸化和含有兩個磷酸化位點的收鈣素蛋白CS-2和(2p)CS-2后,抑制效果隨著添加濃度的升高,成核所需的誘導時間顯著延長,且(2p)CS-2對COM的成核抑制效果更為明顯。通過掃描電鏡及XRD等對成核后的晶體形貌及結(jié)晶性進行檢測,結(jié)果表明CS-2和(2p)CS-2對成核晶體形貌均有影響,且(2p)CS-2可以明顯削弱晶體結(jié)晶性,并有參與成鍵的可能性。(3)借助原位原子力顯微鏡,觀測在不同本底電解質(zhì)的過飽和溶液(σCOM=0.816,I=0.15 mol·L-1,p H 7.0,25°C)中,磷酸化CS-2多肽對COM(-101)和(010)面臺階生長的影響,結(jié)果表明,以Na Cl作為本底電解質(zhì)時,Na Cl對COM晶面臺階的生長具有干擾作用,不利于添加CS多肽后數(shù)據(jù)的測量,因此在AFM實驗中選用KCl調(diào)節(jié)溶液的離子強度。CS-2和(2p)CS-2阻止了臺階的移動,隨著多肽濃度的升高,臺階生長生長速度降低。CS-2對COM(-101)面的抑制效果強于(2p)CS-2,且在相對較高濃度下(≥30 nmol·L-1),臺階生長完全停滯。對于COM(010)面,(2p)CS-2抑制作用較強,尤其對(010)面的[0-2-1]方向。因此,肽段的組成及空間結(jié)構(gòu)決定著它們在不同晶面不同方向臺階上的選擇性吸附。
[Abstract]:Biomineralization is the process of the formation of inorganic solid minerals under the participation and regulation of organic matrix. In this process, the addition of protein molecules can effectively promote or inhibit the inorganic process of organisms. In this study, calcium oxalate monohydrate (COM) crystals, which can be used in atomic force microscope (AFM) (AFM) experiments, were first synthesized by gel method, and then different levels of phosphorylation of cadherin (CS) polypeptides were synthesized in vitro. The effects of cadherin on the nucleation and surface growth of COM crystal were simulated by means of calcium ion electrode detection and in situ atomic force microscopy (AFM). The main results were as follows: (1) silica gel and bentonite were used as culture medium. The results of COM crystal synthesis by gel method showed that the concentration of Ca2 and C _ 2O _ 4 _ 2- was 0.5 mol L ~ (-1) in silica gel system and cultured at room temperature for 60 days. In bentonite system, the crystal structure of COM was stable and the surface was smooth when the concentration of Ca 2 and C 2O 4 2- was 0.75 mol L-1 for 90 days at room temperature and 60 days at 62 鈩，

本文編號：2169640